Seroprevalence and risk factors of Toxoplasma gondii in sheep and goats of North West Province, South Africa

Background The protozoan parasite Toxoplasma gondii causes toxoplasmosis, one of the most prevalent parasitic zoonotic diseases with significant economic and public health implications worldwide. Infection with the parasite has a significant adverse effect on sheep and goat production and can frequently go undetected in the herd, resulting in abortions and weak or dead offspring. Although there are few studies on seroprevalence and risk factors associated with T. gondii infections in livestock in other provinces of South Africa, there is no data in the North West province. Therefore, a cross-sectional study was conducted to investigate the seroprevalence of T. gondii and risk factors associated with exposure in sheep and goats of the North West province of South Africa. Sera from 439 livestock (164 sheep and 285 goats) were collected and analysed for the presence of T. gondii IgG antibodies using indirect ELISA (Enzyme-linked immunosorbent assay). An assessment of potential risk factors in farms associated with seropositivity was also conducted using a structured questionnaire. Results Out of the 439 tested sheep and goats, 13.9% (61/439) were positive for IgG antibodies against T. gondii. Sheep and goats had seroprevalences of 19.5% (32/164) and 10.5% (29/275) respectively. In the multivariable logistic regression model, the risk of acquiring T. gondii was significantly higher in the mixed breed [Odds ratio (OR) = 71.07; 95% confidence interval (CI): 266.8-1893.1; p < 0.011)] animals than white dorper sheep and in farms that burn or bury aborted material (OR = 42.04; CI: 179.9-982.5; p = 0.020) compared to those that only burn aborted material. The risk was lower for the farms in Kagisano-Molopo (OR = 0.00; CI: 0.0-25.4; p = 0.015) and Mahikeng (OR = 0.00; CI: 0.0-4.9; p < 0.001) local municipalities than Greater Taung local municipality, and for the animals that drink water from dams (OR = 0.03; CI: 0.2–58.8; p = 0.021) than those that drink from boreholes. Conclusion The seroprevalence and risk factors associated with transmission observed show that T. gondii infection is widespread in sheep and goats of the North West province. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-024-03939-7.


Background
Toxoplasma gondii (T.gondii), is a ubiquitous obligate intracellular protozoan parasite that causes toxoplasmosis in warm-blooded animals, including humans.The parasite is the only species of the Toxoplasma genus and is a member of the Apicomplex phylum, Conoidasida class, Eucoccidiorida order, and Sarcocystidae family [1].Although many animal species serve as intermediate hosts, only domestic cats, and other felids are definitive hosts and the only species that shed T. gondii oocysts into the environment which makes them important in the epidemiology of the parasite [1][2][3].Both animals and humans can become infected through consumption of food sources containing tissue cysts or contaminated with oocysts [4].Even though infection with T. gondii may show no clinical symptoms, in some intermediate hosts, toxoplasmosis may result in huge economic losses due to reproductive disorders such as abortions and stillbirths [3,5,6].In sheep and goats, toxoplasmosis manifests as a disease of pregnancy, resulting in foetal resorption, stillbirth, mummified lambs, and neonatal mortality [7][8][9].
Although the disease is mostly asymptomatic or only show mild clinical signs in healthy individuals, it may result in miscarriages, stillbirths, and even death in individuals with compromised immune systems, such as those with human immunodeficiency virus (HIV) [10,11].Toxoplasmosis is prevalent in both animals and humans worldwide, including South Africa, and results in huge economic losses in sheep and goats and has serious public health implications which results in congenital defects and ocular diseases in humans [12][13][14][15][16].According to a 2017 study conducted in South Australia by Ryan O'Handley, the cost of sheep toxoplasmosis was estimated to be 70 million Australian dollars, as per a recent ABC news report (https://www.abc.net.au/news/rural/2017-02-07/toxoplasmosis-costs-south-australiansheep-producers/8245676, accessed on 10/01/2024).In another study conducted by Bennett and Ijpelaar in Britain in 2005, they estimated the annual number of affected sheep to be about 334 000, with disease effects of 9.1 million pounds because of abortion or stillbirth and 3.2 million pounds for disease control [17].Despite this, it is still one of the understudied zoonoses with few reports on seroprevalence and risk factors associated with transmission in livestock in South Africa.
A seroprevalence of 6.0%, 2.7%, 6.3%, and 8% in sheep of Gauteng, Free State, KwaZulu-Natal, and Western Cape, respectively was reported in the year 2007 [18,19].In the year 2020, a seroprevalence of 15.2% was also reported in cattle of the Mnisi community in Mpumalanga province [4], and 20.8% in cattle from the high throughput Klerksdorp and Rustenburg abattoirs of the North West province [20].A more recent study conducted in the Eastern Cape province reported a higher seroprevalence in sheep at 64.46%, followed by goats at 53.91%, pigs at 33.90%, chickens at 33.58%, and cats at 32.11% [13].
A study on risk factors associated with T. gondii infection in domestic animals by Tagwireyi [13] found, age, animal production system, cat faeces and feed disposal, climate, location, rodent control, and seropositive cats to be risk factors associated with increased seropositivity of T. gondii for sheep, goats, chicken, and pigs in the Eastern Cape.It is important to control T. gondii infection in sheep and goats not only for reproduction purposes but also for public health reasons.In this current study, we report seroprevalence and risk factors associated with T. gondii exposure in sheep and goats in the North West province of South Africa.

Study area
A cross-sectional study was conducted in the North West province, which is in the northern part of South Africa near the Botswana border and bordered by the Free State to the south, the East Rand to the east, and the Kalahari Desert to the west.It has a population of 3,748 436 people and a 104 882 km² area.Most of the province is made up of flat, sparsely vegetated plains.The province is organized into 18 local municipalities from its four district municipalities.Samples were collected in all four districts (Fig. 1).Villages were selected randomly from each local municipality.In each village, efforts were made to consider all directions (East, West, North, and South of the village using an abstract transect) to avoid bias.A convenient sampling was used to select farmers, and only farmers who gave consent to participate in the study were included.On the farm level, only healthy adult animals and female animals with a history of abortion as pointed out by the farmer were conveniently included in the study.

Sample size and study population
To estimate the sample size for this study, a formula by [21]: n = [( 1.96) 2 P exp(1 − P exp )] /d 2 [21] was used, where p is the estimated prevalence, d the estimated precision, and n the estimated sample size.Due to the lack of data on T. gondii prevalence in the study population, an expected prevalence (P exp) of 50% was used and 5% was set as the estimated precision (d), giving a required minimum sample size of 384.However, for the study, 439 (164 sheep and 275 goats) animals were sampled.

Questionnaire survey
A structured questionnaire was used to determine the risk factors that play role in the exposure of the animals to T. gondii infections.The information collected from questionnaire included age, breed, sex, the origin of the animals (local market, local market and auction, local market and own breed, own breed), location of the farms (district municipality, local municipality), type of farm (communal, commercial), type of breeding (natural, artificial insemination), history of abortion, disposal of aborted material (dispose of, dispose in a hole and close, do not dispose of, used as fertilizer for plants), type of feeding (free grazing, free grazing and farm fed, free grazing and home fed, home fed), feed storage (not applicable, stored in a car garage, stored in a designated room, stored in a designated shack), disposal of manure (dispose of, dispose of in a hole and close, do not dispose of, use as fertilizer for plants), water source (borehole, borehole and dam, borehole and tap water, borehole and river, dam, tap water) and presence of cats in the farms.

Sample collection
Venous blood was collected using BD-Vacutainer® SST™ II Advance 10 ml serum collection tubes.The blood samples were kept in a cooler with ice packs and transported to the laboratory.Sera was harvested the same day by centrifuging the clotted blood at 1000 x g for 10 min and transferred into collection tubes.Sera was stored at -20 °C until analysis.

Serological assay
The sera were used to detect the T. gondii IgG antibodies using the IDEXX T. gondii 2/strip antibody test kit following the manufacturer's instructions (IDDEX Laboratories, Liebelfld-Bern, Switzerland).Frozen sera and reagents from the test kit were brought to room temperature and concentrated wash buffer was diluted 10 times with distilled water.The diluted buffer was used to dilute the positive, and negative controls, as well sera 400-fold.A volume of 100 µl of the diluted controls and sera was transferred into the T. gondii antigen-coated plate wells and incubated for an hour at 37 °C.The plate was washed three times with 300 µl of the diluted wash buffer after incubation.The wash buffer residues were removed by gently tapping the plate on absorbent paper.A 100 µl of the conjugate was added to each well and incubated for an hour at 37 °C.The plates were again washed three times with 300 µl wash buffer with wash buffer residues removed as described above, and 100 µl of 3, 30, 5, 50-Tetramethylbenzidine (TMB) was added and incubated for 15 min in the dark at room temperature.The stop solution of 100 µl to was then added to each well following incubation.The absorbance at 450 nanometres was immediately measured using a Thermo Labsystems MultiskanMS Original microplate reader (Thermo Fischer Scientific, Waltham, MA, USA).
The assay validity was evaluated using the following criteria: The two negative controls average (NCx) optical density value at 450 nm (A450) should be less than or equal to (≥) 0.500 to determine the validity of the test.At 450 nm, the two positive controls average value (PCx) should be ≤ 2.500 and the PCx-NCx should be larger than or equal to (≤) 0.300.The following formula was used to determine sample to positive (S/P) ratio: For results interpretation, S/P % <20 represented a negative result, 20 ≤ S/P % <30 represented a suspect, 30 ≤ S/P% <100 represented a weak positive result, and S/P % ≥100 represented a positive result.

Data analysis
The data was captured and cleaned using a Microsoft Excel spreadsheet and analysed using Stata 14.0 (Stata-Corp, College Station, Texas, USA).To determine the association between the potential risk factors and seropositivity of T. gondii, a univariate analysis using a 2 × 2 contingency table was done and a Chi-square test with appropriate p-value was reported.Chi-square test P ≤ 0.2 was used as an inclusion criterion in the multiple logistic regression model as recommended by [22].Multivariable analysis was done using multilevel logistic regression models and a stepwise procedure was performed by removing variables with P > 0.05.The Akaike's secondorder information criterion (AIC) was calculated for each model, and the process was repeated until the model with the lowest AIC was identified.Results for the odds ratios with a 95% confidence interval were reported.

Discussion
For the first time in the North West province of South Africa, we determined the seroprevalence of T. gondii in sheep and goats, using an indirect ELISA.Overall, we found a seroprevalence of 13.9%, with sheep (19.5%) having a higher seroprevalence than goats (10.5%).The difference in seroprevalence between the two species could be attributed to the fact that goats are natural browsers that eat leaves and twigs from taller bushes and shrubs, making them less likely to ingest oocysts, whereas sheep are grazers who eat short grasses and clovers that are near the soil [23][24][25].The overall seroprevalence reported in this study is lower than the 67.2% reported in Zimbabwe for sheep and goats [26], and the 64.5% and 53.9% for sheep and goats respectively, reported in the Eastern Cape in the year 2019 [13].This pronounced higher differences could be attributed to the climatic variations between North West and Eastern Cape province (dry and semidesert vs. moist and humid), since the moist and humid environment is conducive for the survival of oocytes and spread compared to dry and semi-desert environment, leading to increased seropositivity [27,28].Seropositivity for goats agreed with the 10% found in Botswana [29].In our study, females had seropositivity of 14.6% against 5.9% in males, a finding similar to the one from the Eastern Cape [13].The difference in seropositivity observed between females and males could be due to hormonal fluctuations, immunosuppression associated with pregnancy, lactation stress, and physiological changes which makes females more susceptible to T. gondii infections than males [30].A significant association between T. gondii seropositivity and breed was observed with mixed breed having the highest seropositivity (18.2%) when compared to other breeds.Inbreeding is known to weaken the genetic makeup of animals, thus making them more prone to diseases than pure breeds, which could have been the case with the higher seropositivity observed in this study [3,31,32].The difference in seropositivity between the animals that were allowed to breed naturally (14.2%) and those that were artificially inseminated (5.9%) is consistent with previous studies on sexual transmission of the parasite using both the methods [3,31,33,34].This might indicate a possible sexual transmission through the natural breeding rather  The presence of cats on the farms was a significant risk factor in this study, which was expected given their role as definitive hosts in the spread of T. gondii [3].This finding was consistent with those that were reported in Romania [35].Differences in seroprevalence among the district and local municipalities played a role in the exposure of the animals to the parasite, a result consistent with that of Eastern Cape, South Africa [13].The different temperatures, humidity, and annual rainfall among the district municipalities could have contributed to the variation of seropositivity as the humidity in the Kagisano-Molopo local municipality and the hot weather in Mahikeng local municipality.These climatic variances agree with the ones found in the different regional states in Central Ethiopia where altitudes, rainfall and temperatures were different among the studied districts [36].Free grazing and home fed animals system had a significant association with higher seropositivity compared to the rest of the feeding systems.These findings further support the role cats play in the epidemiology of the parasite as they could have potentially contaminated the feed of the animals [37][38][39].Disposal of manure was also a significant factor with the highest seropositivity observed from farms that uses manure as a fertilizer.This significance is no surprise particularly when factoring in the presence of cats in the farms, an indication of shedding oocysts on the feed, soil, pastures, manure, and water sources of the animals which increases their risk of infection [16,40].The introduction of diseases that have not previously existed or have been eradicated in a herd is highly influenced by the origin of newly introduced animals.This was the case even with the significant finding in this study, which demonstrated seropositivity rates of 62.2% on farms that purchase animals from local markets and auctions, compared to those only originating from auctions, indicating a potential for exposure as reported in the Eastern Cape province [13].The animals with a history of abortion were more susceptible to exposure than the ones that did not have (23.8% vs. 11.7%).A study in Botswana reported similar findings from animals with a history of abortion [29].Animals from farms that burn or burry aborted material had the highest seropositivity of 33.3% than animals from farms that send aborted material to sate vet (0%), which demonstrates the importance of proper disposal of aborted material in preventing the spread of T. gondii as pets can get infected by eating the aborted material containing T. gondii tissue cysts or tachyzoites [41,42].This significance corroborates previous findings where high seropositivity was found in farms that bury or feed the aborted material to pets [43,44].
According to the final multivariable logistic model, mixed-breed Dorpers had a higher risk of acquiring an infection than white Dorpers, which is most likely because of the subpar hygienic-sanitary practices used to maintain mixed-breed livestock on farms, as was noted in a related study from mixed-breed in Northern Italy [45] [2, 13, 36].Although a study conducted in the north-eastern areas of Colombia found that small ruminants that drink from a dam have a lower chance of being infected, our study indicates that long-standing water can get contaminated with oocytes, thus acting as a source of infection for livestock compared to boreholes [46].

Conclusion
This study showed the seroprevalence and risk factors associated with exposure of sheep and goats to T. gondii infections for the first time in the North West province, South Africa.These findings further demonstrate the need for surveillance of T. gondii together with the rest of the reproductive pathogens in sheep and goats and should not be neglected during abortion or stillbirth cases.

Limitation
Due to the lack of consent from the farmers in the Namusa, Lekwa-Teema, Tswaing, Ditsobotla, and Matlosana local municipalities, these municipalities were not included in the study.This was a limitation as the data obtained does not represent them.

Fig. 1
Fig. 1 Map of North West province districts and local municipalities where sampling was conducted

Table 1
Univariate risk analysis of factors associated with T. gondii seropositivity in sheep and goats than contamination from non-sterile artificial insemination apparatus and procedures.